Mitotic CDK1 and 4E-BP1 I: Loss of 4E-BP1 serine 82 phosphorylation promotes proliferative polycystic disease and lymphoma in aged or sublethally irradiated mice

4E-BP1 is a tumor suppressor regulating cap-dependent translation that is in turn controlled by mechanistic target of rapamycin (mTOR) or cyclin-dependent kinase 1 (CDK1) phosphorylation. 4E-BP1 serine 82 (S82) is phosphorylated by CDK1, but not mTOR, and the consequences of this mitosis-specific phosphorylation are unknown. Knock-in mice were generated with a single 4E-BP1 S82 alanine (S82A) substitution leaving other phosphorylation sites intact. S82A mice were fertile and exhibited no gross developmental or behavioral abnormalities, but the homozygotes developed diffuse and severe polycystic liver and kidney disease with aging, and lymphoid malignancies after irradiation. Sublethal irradiation caused immature T-cell lymphoma only in S82A mice while S82A homozygous mice have normal T-cell hematopoiesis before irradiation. Whole genome sequencing identified PTEN mutations in S82A lymphoma and impaired PTEN expression was verified in S82A lymphomas derived cell lines. Our study suggests that the absence of 4E-BP1S82 phosphorylation, a subtle change in 4E-BP1 phosphorylation, might predispose to polycystic proliferative disease and lymphoma under certain stressful circumstances, such as aging and irradiation.

To study the function of 4E-BP1 S82 phosphorylation, we established homozygous S82 alanine substitution (S82A) knock-in C57/B6N mice. Aged homozygous S82A mice developed degenerative polycystic kidney and liver disease not present in wild-type littermates. On 9 Gy total body irradiation, S82A mice developed T cell lymphomas that were not present in irradiated wild-type littermates. These phenotypes are remarkable given that other phosphorylation sites in 4E-BP1 were functionally intact [18] and mice in which the entire Eif4ebp1 gene is deleted remain viable [22]. Our study suggests that the absence of 4E-BP1 S82 phosphorylation by CDK1, effected by a single amino acid substitution from S82 to alanine, can predispose to polycystic proliferative disease and lymphoma.

Results
Aged S82A mice are prone to polycystic liver and kidney disease and lymphoma 4E-BP1 S82A knock-in mice were generated using a Cre-loxP cloning strategy (S1A Fig). A hemizygous founder was used to generate heterozygous 4E-BP1 S82A mice, which in turn were used to generate the homozygous Eif4ebp1 S82A/S82A (S82A) and Eif4ebp1 WT/WT (WT) littermate mice used in this study. The generation of a single point mutation was confirmed by genotyping PCR, immunoblotting and whole genome sequencing (WGS) (S1B-S1D Fig).
Littermate S82A and WT mice were fertile and exhibited no gross developmental or behavioral abnormalities.
Overall survival did not significantly differ between S82A and WT mice ( Fig 1A). Tumor masses were detected in eleven of twenty-eight (39.3%) S82A mice and in eight of twentythree (34.8%) WT mice (Fig 1B). Eight of twenty-eight (28.6%) S82A mice developed lymphocytic lymphomas compared to four of twenty-three (17.4%, p = 0.51 FET) WT mice (Table 1 and Fig 1A). Seven of the eight S82A lymphomas were of CD19+ B cell origin, as were all four of the WT lymphomas. Other sporadic cancers, including angiosarcoma and adenocarcinoma, did not significantly differ between the two mouse strains (Table 1). No splenomegaly or lymphadenopathy was detectable on gross examination in the S82A mouse strain. . S82A mice (n = 31, red line) and WT mice (n = 24, black line) were subjected to long-term observation. Mice with 20% weight loss or illness were euthanized and processed for gross anatomic and histologic examination. Log-rank (Mantel-Cox) test was used to compare survival probability. N.S. indicates difference is not significant. 11 of 28 (39.3%) S82A mice and 8 of 23 (34.8%) WT mice died with observable lesions. Chronologic occurrence of lesions are marked with grey arrows (solid for S82A and dashed for WT). Column plot (right panel) summarizes the percentage of mice with tumors (grey shading) found in S82A and WT and mice. (B) S82A mice aging mice develop polycystic liver and kidney lesions. On gross examination, S82A livers in some mice were enlarged by variably-sized cysts containing clear to yellow fluid affecting all lobes. These areas correspond microscopically to loss of hepatic parenchyma and replacement by cystic spaces lined by cuboidal epithelium within a fibrous connective tissue stroma containing lymphocytic infiltrates. S82A kidneys with cystic lesions microscopically had elongated cysts of varying sizes within the renal cortex. Cysts were lined by cuboidal to attenuated epithelial cells (Top row). Cystic lesions were not found in WT animals (Bottom row).
https://doi.org/10.1371/journal.pone.0282722.g001  S82A mice also showed extensive non-neoplastic liver and kidney disease (Fig 1B), characterized by polycystic changes affecting variable proportions of the organ parenchyma, that did not have an overall impact on survival. Mild to moderate lymphocytic infiltrates were present within the fibrous stroma surrounding cysts. Liver polycystic disease (LPD), frequently severe with near-complete effacement of healthy liver tissue, occurred in eleven of twenty-eight S82A mice, (39.3%). Microscopically, hepatic cystic structures were lined by cuboidal epithelium consistent with bile duct origin. Cystic changes were also seen in kidneys for five (17.9%) S82A mice. Microscopically, renal cysts were lined by cuboidal to attenuated epithelial cells. All mice with polycystic changes in kidneys also had polycystic changes in the liver. No cystic changes were seen in either the liver or the kidneys among control WT littermates on either gross or microscopic examination. Immunoblotting of lysates made from various organs and tissues showed that 4E-BP1 expression levels in S82A homozygous mice was comparable to WT mice formally confirming that there is no disruption of 4E-BP1 expression by the generation of the knock-in model. However, 4E-BP1 S82 phosphorylation deficiency appeared to lead to a more hyperphosphorylated 4E-BP1 profile specifically in liver tissue from S82A homozygous mice (S2 Fig).

Sublethally irradiated S82A mice are prone to immature T-cell lymphoma with Pten-mutation
To determine if neoplastic changes in S82A mice could be accelerated by stressors such as ionizing radiation (IR), eleven to thirteen-week-old S82A and WT mice were subjected to 9 Gy total body irradiation (TBI), the approximate LD 50 for C57BL/6N mice in our irradiator facility. Twelve of twenty-one (57.1%) of WT and twenty of twenty-seven (74.1%) S82A mice died from radiation toxicity within 5 weeks post-irradiation, a difference that was not statistically significant (S3A Fig). Similarly, there was no significant difference intestinal crypt stem cell regeneration after exposure to 15 Gy TBI [23], suggesting that these cells from S82A mice were not specifically radiosensitive (S3B Fig). However, of the seven S82A mice surviving acute 9 Gy TBI radiation toxicity, five (71%) developed lymphomas at 20-47 weeks post-irradiation while none of the surviving eight irradiated WT mice developed lymphoma (Table 2 and Fig  2A). Two additional S82A mice became moribund (defined as >20% body weight loss) and were euthanized but no tumors were found at necropsy. One of these mice exhibited cystic liver disease.
Four tumors originating in irradiated S82A mice were examined and found to have CD3+ and CD8+ or CD4+/CD8+ (double positive, DP) T cell markers, consistent with immature T-cell lymphomas (Fig 2B). The fifth S82A tumor was necrotic and could not be immunophenotyped. Among mice surviving 9 Gy TBI, S82A mice had significantly reduced long-term survival (after recovery from acute effects) compared to WT mice (Log-rank test, p = 0.016) (Fig 2A). The T cell origin of lymphomas developing in irradiated mice contrasts with the B cell lymphomas developing in non-irradiated mice.
To determine if hematopoietic stem/progenitor cell (HSPC) populations differ between S82A and WT mice, bone marrow HSPC were harvested but no significant differences between LSK (Lin -/Sca-1 + /c-Kit + ) or myeloid progenitor LK (Lin -/Sca-1 -/c-Kit + ) cell populations were seen (S4A To identify driver mutations in S82A lymphomas, whole genome sequencing was performed on 3 lymphoma and 1 non-irradiated S82A brain tissue and 26 lymphoma-related mutations were identified (Fig 2C). Seven gene mutations (Ankrd11, C130026I21 Rik, Gm10717, Gm10721, Gm11168, Muc4, and Zfp984) were hetero-/hemi-zygous in all three lymphomas and one gene (Itgav) had homozygous mutations in one lymphoma. Two lymphomas contained mutations in Pten, a common lymphoma driver gene: one contained a missense mutation, and another contained a splice site mutation. Cell lines were established from two lymphomas arising in the irradiated S82A mice (Fig 2D and Table 2) and had reduced or absent PTEN expression compared to control thymocytes isolated from S82A and WT mice (Fig 2D), suggesting irradiated S82A mice are more prone to PTEN mutation.

Discussion
Since cell culture models have not revealed a physiological function for phosphorylation of the 4E-BP1 S82 residue [18,21], we sought to examine its role in mice in which the S82 residue alone is mutated to a non-phosphorylated alanine. These unstressed, homozygous mice were   WT mice (n = 9, black line) and S82A mice (n = 7, red line) that survived the acute death phase were followed for long-term observation. Log-rank (Mantel-Cox) test was used to compare survival probability. Significance is indicated with an asterisk * (p = 0.016). Mice dying with tumors are marked with generally healthy, fertile and had similar lifespans to wild-type littermate control mice, consistent with all other 4E-BP1 regulatory sites (e.g., T37, T46, T70, and S65) being intact and regulated by mTOR and CDK1 in a similar manner to the wild-type protein. The S82A knock-in mutation resulted in tissue-specific pathologies, particularly B and T cell lymphomagenesis and liver/kidney polycystic disease that was only evident at necropsy. These effects were markedly accelerated after recovery from the stress of TBI, leading to premature mortality in the S82A homozygotes. It has been shown that intra-thymic T cells can be relatively resistant and auto-reconstitute after irradiation unlike T cells from other hematopoietic and lymphoid organs [24]. This may potentially explain the T cell lymphoma phenotype in S82A homozygous mice. Previous studies have reported Trp53(p53) knockout mice to spontaneously develop immature T cell lymphomas in their first six months of life with all tumour samples having lost or reduced Pten [25]. We found similar Pten results in 4e-bp1 S82A T cell lymphoma samples (Fig 2D). The critical role of Pten in preventing T cell lymphomagenesis has been well documented and studied [26]. However, we did not find apparent p53 mutation or deletion in 4e-bp1 S82A tumour samples. Meanwhile, we found intact Pten expression and normal T cell development in young 4e-bp1 S82A mice, which suggests that additional stress stimuli are essential for 4e-bp1 S82A mice lymphomagenesis. 4e-bp1 S82A mice may be better able to retain and allow expansion of mutated cells resulting from aging or sublethal irradiation.
Polycystic kidney disease (PKD) has been described as a "neoplasia in disguise" [27] that may be related to perturbed mTOR signaling [28]. PKD is the most common hereditary cause of kidney failure and is commonly associated with liver cystic liver changes [29]. Holditch and colleagues found elevated 4E-BP1 hyperphosphorylation in human PKD human tissues and kidneys from PKD-prone mice [30]. PKD disease severity in PKD mice, however, was unexpectedly increased after viral vector transduction of mTOR-resistant 4E-BP1 F113A consistent with 4E-BP1 dysregulation, rather than inhibition, contributing to PKD. Recent studies also point towards CDK1 dysfunction predisposing to cystic organ disease [31], which is supported by our study. Since cystic disease was mainly evident in aging S82A mice, our data suggests that 4E-BP1 S82 phosphorylation by CDK1 may be critical for tissue renewal of differentiated cell populations rather than progenitor cell populations involved in organogenesis.
Even though we did not find clear mechanisms to account for the phenotype observed in the 4E-BP1 S82A mouse, this study provides some intriguing data suggesting that 4E-BP1 S82 phosphorylation is beneficial for overcoming some cellular stress conditions such as aging and irradiation. Several important caveats should be considered in interpreting our data. It is possible that 4E-BP1 S82 phosphorylation is critical to a tissue or specific cell type more pertinent to the phenotype that we did not sample in our studies (e.g., liver or kidney cells; B cell; other T cell populations). Although markers such as CD34, cytoplasmic CD3 or deoxynucleotidyl transferase [32] may help define the origin and identity of the immature T-cell lymphomas in our study, we did not perform this immuno-profiling on the lymphoma grey arrows. Column plot (right panel) summarizes the percentage of S82A and WT found with tumors. (B) Immunohistochemical confirmation of T-cell tumors isolated from mediastinal regions of two S82A mice (#1 and #5). Tumors were immunostained with CD3, CD4, CD8 and CD19 antibody. Brown signal indicates positive reactivity with respective antibodies. Images were captured at 40x magnification. (C) Common mutations identified in at least two 4E-BP1 S82A lymphoma tissues (#2, #3, and #4) by Oncoplots. Types of mutations are color-coded, and zygosity of mutations: heterozygous (-/+), homozygous (+/+), and not applicable (N.A.) are indicated for each mutations identified. (D) CD4 and CD8 staining profiles for T-cell lymphoma (TCL) cell lines (Left), #5 and #3 were established from two different tumor explants. Immunoblotting for T-cell lymphoma (TCL) cell lines (Right). Thymocytes were acquired from 4-week-old WT and S82A mice. Total cell lysates were used for immunoblotting with indicated antibodies.
https://doi.org/10.1371/journal.pone.0282722.g002 samples. We did not track lymphomagenic capacity after irradiation, and perhaps in the T cell population this may have revealed differences in regeneration between the S82A and WT mice. Since the observed phenotype in aged S82A mice develops very late, mating this S82A mouse strain with an aging mouse model strain might reproduce the observed phenotype at an earlier age.
Nevertheless, despite these caveats, this mouse strain has utility in separating the effects of CDK1 from mTOR on 4E-BP1 function and provides a new model for polycystic organ disease that is open to therapeutic interventions.

Ethics statement
Mice breeding and long-term monitoring experiment was approved by the Institutional Animal Care and Use Committee (IACUC) guidelines and were approved by the Animal Ethics Committee at University of Pittsburgh (IACUC breeding protocol #18011983, IACUC experimental protocol #18012088, and TBI protocols #18022000 and #18073023). Per protocol, mice were sacrificed by using 100% CO 2 followed by cervical dislocation if mice developed tumors greater than 1.8 cm in diameter, or if the mice showed any signs of persistent morbidity such as loss in weight greater than 20%, lethargy, unwillingness to ambulate, hunched posturing and ruffled fur. No invasive procedures likely to produce moderate to severe pain were performed; 3-5% isoflurane inhalant induction and maintenance was performed for genotyping studies. All efforts were made to minimize possible pain and suffering for mice during irradiation, monitoring, and euthanasia.

Generation of 4E-BP1 S82A (S82A) knock-in mouse
4E-BP1 Ser(S)83 phosphorylation is evolutionally conserved in vertebrates and the human 4E-BP1 S83 residue corresponds to mouse 4E-BP1 S82 (Fig 1A). To study the physiological function of 4E-BP1 S83 phosphorylation, knock-in mice were generated commercially (Gen-O-Way) that express phosphorylation-defective 4E-BP1 containing S82 AGC -to-Ala(A)82 GCT (S82A) mutation. Briefly, 4E-BP1 S82A allele was introduced to the mouse embryonic stem (ES) cell genome through homologous recombination. The successfully targeted ES cells were verified by Southern hybridization (S1B Fig) and injected into blastocysts to develop the chimeric mice. The chimeric male mice were mated with C57BL/6N Cre female mice to excise loxP-Neo cassette. Mice harboring germline-transmitted 4E-BP1 S82A allele were selected as heterozygous founders. S82A homozygous mice were established by heterozygous breeding. Homozygous and heterozygous S82A mice were fertile and not obviously different from WT mice in Mendelian sex ratios, perinatal mortality or weight gain.

Mouse experiments
For long-term observation, mouse body weight was monitored every week. Mice with 20% body weight loss or compromised body conditions were euthanized. Tissues were harvested, formalin-fixed, and embedded in paraffin blocks (FFPE). For irradiation experiments, 11~13-week-old mice were subjected to total body irradiation at 9 Gy. Mice survived from acute irradiation syndrome were monitored every week. Euthanasia criteria are the same as in long-term observation mice. Survival curves were made with Prism GraphPad.

Thymocytes and splenocytes isolation
Spleen and thymus from 4~8-week-old mice were harvested in R10 media (RPMI-1640 supplemented with 10% FBS, non-essential amino acid, sodium pyruvate, 100 U/ml Penicillin/ Streptomycin, 20 mM HEPES and 50 μM β-ME) on ice. Collected tissues were ground between frosted glass slides and filtered through 70 μm filter. Pelleted cells were resuspended in 1 ml RBC lysis buffer (0.15 M NH 4 Cl, 10 mM NaHCO 3 , 0.1 mM EDTA, pH 8.0) for 30-40 seconds and diluted with 4 ml serum free media immediately after lysis. Cells were washed with R10 media and filtered through 70 μm filter for downstream experiment.

Whole genome sequencing and mutation analysis
Paired end Illumina sequencing reads were checked for quality and mapped to the mouse genome (mm10) using the DNA Nexus platform's pipeline. Mutect (v1.1.7) and Varscan2 (v2.4.2) was used for identifying somatic variants. Variants were annotated using Vcf2maf (v1.6.16) and Variant Effect Predictor (VEP v95) to produce mutation annotation format (MAF) files. A union of annotated variants from Mutect and Varscan2 was used for further analysis. Oncoplots were generated using maftools (v2.2.0). The zygosity is estimated by the variant calling tools.